The endocytic pathway )EP) is central to the function of amyloid precursor protein (APP), Abeta, apolipoprotein E and other molecules etiologically linked to Alzheimer's disease (AD) pathogenesis. Recently, we identified abnormalities of early endosomes (EE), implying strong EP activation, in control and hippocampal neurons before beta-amyloid deposition in sporadic AD (SAD) and Down Syndrome (DS). The overall objective of this project is to define the consequences of EP abnormalities for Abeta production, Abeta clearance, and cell survival in SAD and the influence of APOE genotype and vascular risk factors on these interrelationships. In well-characterized AD brains at the APOE epsilon allele accentuates the onset and magnitude of EE abnormalities (Aim 1). Based on preliminary data, we will investigate the localization and increased context of Abeta in EE from SAD and early DS brain using ICC and confocal microscopy on tissues and on purified EE fractions using immunoblot assays (Aim 1). Based on preliminary data, we will investigate the localization and increased content of Abeta in EE from SAD and early DS brain using ICC and confocal microscopy on tissues and on purified EE fractions using immunoblot assays (Aim 1) and immuno-EM. Changes in the protease composition of EE will also be analyzed based on our earlier findings of increased lysosomal hydrolase expression and trafficking to EE in AD. In Aim 2, we will model the abnormalities seen in SAD and DS by appropriate genetic manipulations that upregulate endocytosis and direct hydrolyases preferentially to EE in transfected cells and transgenic mice to test predicted effects of these SAD mechanisms on Abeta biology and cell survival. In Aim 3, the evolution of these EPO abnormalities will be investigated in mice trisomic for a segment of the human homolog of chromosome 21 where we have seen EP disturbances, including Abeta accumulation in EE, which are similar to those in mice expressing human APOE alleles. In aim 4, neuron-vascular interrelationships suggested by the presence of EP and lysosomal system activation in cerebrovascular endothelia will be investigated by characterizing these abnormalities in SAD, inherited cerebral amyloid angiopathy (CAA), and animal and cultured human endothelial cell models of CAA and ischemia. This project will clarify the hypothesized role of EP abnormalities, the earliest known cellular response in SAD, in accelerating amyloidogenesis as well as generate novel models of SAD-relevant pathology and identify new targets for possible therapeutic intervention at stages of disease well before clinical onset.